Conservation and divergence in cellulosome architecture between two strains of Ruminococcus flavefaciens

A 17-kb scaffoldin gene cluster in Ruminococcus flavefaciens strain FD-1 was compared with the homologous segment published for strain 17. Although the general design of the cluster is identical in the two strains, significant differences in the modular architecture of the scaffoldin proteins were discovered, implying strain-specific divergence in cellulosome organization.     

Segmental duplication associated with evolutionary instability of human chromosome 3p25.1

Fluorescence in situ hybridization (FISH) of human bacterial artificial chromosome (BAC) clones to orangutan metaphase spreads localized a breakpoint between human chromosome 3p25.1 and orangutan chromosome 2 to a <30-kb interval. The inversion occurred in a relatively gene-rich region with seven genes within 500 kb. The underlying breakpoint is closely juxtaposed to validated genes, however no functional gene has been disrupted by the evolutionary rearrangement. An approximately 21-kb DNA segment at the 3p25.1 breakpoint region has been duplicated intrachromosomally and interchromosomally to multiple regions in the orangutan and human genomes, providing additional evidence for the role of segmental duplications in hominoid chromosome evolution.     

Molecular linkage of the HLA-DR, HLA-DQ, and HLA-DO genes in yeast artificial chromosomes.

Eight major histocompatibility complex (MHC) class II loci and the newly defined Y3/Ring 4 locus were isolated in overlapping yeast artificial chromosome (YAC) clones defining a 420-kb segment of human chromosome 6p21.3. YAC B1D12 spanning 320 kb contained seven of these loci from HLA-DRA to HLA-DQB2. A 330-kb YAC, A148A7, spanned from the HLA-DQA1 locus through the Y3/Ring 4 locus and extended at least 130 kb centromeric of YAC B1D12. Southern blotting demonstrated that YAC B1D12 derived from the HLA-DR3 haplotype and that YAC A148A7 derived from the HLA-DR7 haplotype of the heterozygous library donor. A third 150-kb YAC, A95C5, lay within this contig and contained only the HLA-DRA locus. A fourth 300-kb YAC, A76F11, was isolated by chromosome walking from the telomeric end of YAC B1D12. Probes isolated from the ends of the YAC genomic inserts have been used to confirm overlaps between the clones. These analyses demonstrated that the centromeric end of YAC A76F11 used the same genomic EcoRI cloning site as the telomeric end of YAC A95C5. YAC B1D12 used an EcoRI site only 2.1 kb telomeric of the aforementioned EcoRI site. These data suggest that certain EcoRI sites are used preferentially during construction of the library. These YACs complete the linkage of the DR and DQ subregions of the HLA complex in cloned DNA and provide the substrate for precise analysis of this portion of the class II region.     

The 102-Kilobase Unstable Region of Yersinia pestis Comprises a High-Pathogenicity Island Linked to a Pigmentation Segment Which Undergoes Internal Rearrangement

Several pathogenicity islands have recently been identified in different bacterial species, including a high-pathogenicity island (HPI) in Yersinia enterocolitica 1B. In Y. pestis, a 102-kb chromosomal fragment (pgm locus) that carries genes involved in iron acquisition and colony pigmentation can be deleted en bloc. In this study, characterization and mapping of the 102-kb region of Y. pestis 6/69 were performed to determine if this unstable region is a pathogenicity island. We found that the 102-kb region of Y. pestis is composed of two clearly distinct regions: an ≈35-kb iron acquisition segment, which is an HPI per se, linked to an ≈68-kb pigmentation segment. This linkage was preserved in all of the Y. pestis strains studied. However, several nonpigmented Y. pestis strains harboring an irp2 gene have been previously identified, suggesting that the pigmentation segment is independently mobile. Comparison of the physical map of the 102-kb region of these strains with that of strain 6/69 and complementation experiments were carried out to determine the genetic basis of this phenomenon. We demonstrate that several different mechanisms involving mutations and various-size deletions are responsible for the nonpigmented phenotype in the nine strains studied. However, no deletion corresponded exactly to the pigmentation segment. The 102-kb region of Y. pestis is an evolutionarily stable linkage of an HPI with a pigmentation segment in a region of the chromosome prone to rearrangement in vitro.     

Mechanisms of sod2 gene amplification in Schizosaccharomyces pombe

Gene amplification in eukaryotes plays an important role in drug resistance, tumorigenesis, and evolution. The Schizosaccharomyces pombe sod2 gene provides a useful model system to analyze this process. sod2 is near the telomere of chromosome I and encodes a plasma membrane Na(+)(Li(+))/H(+) antiporter. When sod2 is amplified, S. pombe survives otherwise lethal concentrations of LiCl, and >90% of the amplified sod2 genes are found in 180- and 225-kilobase (kb) linear amplicons. The sequence of the novel joint of the 180-kb amplicon indicates that it is formed by recombination between homologous regions near the telomeres of the long arm of chromosome I and the short arm of chromosome II. The 225-kb amplicon, isolated three times more frequently than the 180-kb amplicon, is a palindrome derived from a region near the telomere of chromosome I. The center of symmetry of this palindrome contains an inverted repeat consisting of two identical 134-base pair sequences separated by a 290-base pair spacer. LiCl-resistant mutants arise 200-600 times more frequently in strains deficient for topoisomerases or DNA ligase activity than in wild-type strains, but the mutant cells contain the same amplicons. These data suggest that amplicon formation may begin with DNA lesions such as breaks. In the case of the 225-kb amplicon, the breaks may lead to a hairpin structure, which is then replicated to form a double-stranded linear amplicon, or to a cruciform structure, which is then resolved to yield the same amplicon.     

Genetic dissection and pyramiding of quantitative traits for panicle architecture by using chromosomal segment substitution lines in rice

To understand the genetic basis of yield-related traits of rice, we developed 39 chromosome segment substitution lines (CSSLs) from a cross between an average-yielding japonica cultivar, Sasanishiki, as the recurrent parent and a high-yielding indica cultivar, Habataki, as the donor. Five morphological components of panicle architecture in the CSSLs were evaluated in 2 years, and 38 quantitative trait loci (QTLs) distributed on 11 chromosomes were detected. The additive effect of each QTL was relatively small, suggesting that none of the QTLs could explain much of the phenotypic difference in sink size between Sasanishiki and Habataki. We developed nearly isogenic lines for two major QTLs, qSBN1 (for secondary branch number on chromosome 1) and qPBN6 (for primary branch number on chromosome 6), and a line containing both. Phenotypic analysis of these lines revealed that qSBN1 and qPBN6 contributed independently to sink size and that the combined line produced more spikelets. This suggests that the cumulative effects of QTLs distributed throughout the genome form the major genetic basis of panicle architecture in rice. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Ando and T. Yamamoto contributed equally to this work.     

Chromosome walking shows a highly homologous repetitive sequence present in all the centromere regions of fission yeast

By cloning centromere-linked genes followed by partial overlapping hybridization, we constructed a 210-kb map encompassing the centromere in chromosome II and a 60-kp map near the centromere of chromosome I in the fission yeast Schizosaccharomyces pombe which has three chromosomes. Integration of the cloned sequences into the chromosome and subsequent analyses of tetrads and dyads revealed an approximately 50 kb long domain located in the middle of the 210-kb map, tightly linked to the centromere and greatly reduced in meiotic recombination. This domain contained at least two classes of repetitive sequences. One, designated yn1, was specifically present in a particular chromosome and repeated three times in the 210-kb map of chromosome II. The other, designated dg, was located in all the centromere regions of three chromosomes. One (dgI) and two (dgIIa, dgIIb) copies of the dg were found in the maps of chromosomes I and II, respectively. The dgIIa and dgIIb were arranged with a 20-kb interval within the repetitive domain. In the centric region of chromosome II, 3-4 copies of the dg appeared to exist. By determining the nucleotide sequences of dgI and dgIIa, the dg was identified to be 3.8 kb long. The sequence homology was 99% between dgI and dgIIa. These extraordinarily homologous sequences seemed not to be transcribed into RNA nor to be encoding any protein. The larger part of the dg sequence was internally non-repetitious, a 600-bp region existed which consisted of stretches of several short repeating units. The structures in or surrounding the centromeres of S. pombe appear to be much more complex than those of the budding yeast Saccharomyces cerevisiae.     

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.     

Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice

Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, Toledo(BAC) virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. Toledo(BAC), like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (Toledo(BAC)) retained its virulence. The 15-kb segment was then deleted from Toledo(BAC), and the resulting virus, Toledo(Delta15kb), was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. Toledo(Delta15kb) had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the Toledo(Delta15kb) genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.     

Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5′-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3′-deleted pyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.     

Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2.

The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth.     

Genetic linkage of the human apolipoprotein AI-CIII-AIV gene cluster and the neural cell adhesion molecule (NCAM) gene

The genes encoding apolipoproteins AI, CIII, and AIV, three plasma proteins involved in lipid metabolism, are clustered within a 15-kb DNA segment (apoAI-CIII-AIV gene cluster) located on human chromosome 11 at band q23. The gene encoding the neural cell adhesion molecule (NCAM), a cell surface glycoprotein involved in cell-cell recognition during morphogenesis, is also located on chromosome 11, band q23. In this report, 12 previously described restriction fragment length polymorphisms (RFLPs) in the apoAI-CIII-AIV gene cluster were tested for cosegregation with a newly identified BamHI RFLP in the NCAM gene using 13 families. The results show that the apoAI-CIII-AIV gene cluster and the NCAM gene loci are linked with a maximum lod score of 15.9 at a recombination fraction of 0.028. In addition, an approach for the most efficient use of the apoAI-CIII-AIV gene cluster polymorphisms, based on the evaluation of their individual and cumulative heterozygosities, is presented.     

TAC1, a major quantitative trait locus controlling tiller angle in rice

A critical step during rice (Oryza sativa) cultivation is dense planting: a wider tiller angle will increase leaf shade and decrease photosynthesis efficiency, whereas a narrower tiller angle makes for more efficient plant architecture. The molecular basis of tiller angle remains unknown. This research demonstrates that tiller angle is controlled by a major quantitative trait locus, TAC1 (Tiller Angle Control 1). TAC1 was mapped to a 35-kb region on chromosome 9 using a large F(2) population from crosses between an indica rice, IR24, which displays a relatively spread-out plant architecture, and an introgressed line, IL55, derived from japonica rice Asominori, which displays a compact plant architecture with extremely erect tillers. Genetic complementation further identified the TAC1 gene, which harbors three introns in its coding region and a fourth 1.5-kb intron in the 3'-untranslated region. A mutation in the 3'-splicing site of this 1.5-kb intron from 'AGGA' to 'GGGA' decreases the level of tac1, resulting in a compact plant architecture with a tiller angle close to zero. Further sequence verification of the mutation in the 3'-splicing site of the 1.5-kb intron revealed that the tac1 mutation 'GGGA' was present in 88 compact japonica rice accessions and TAC1 with 'AGGA' was present in 21 wild rice accessions and 43 indica rice accessions, all with the spread-out form, indicating that tac1 had been extensively utilized in densely planted rice grown in high-latitude temperate areas and at high altitudes where japonica rice varieties are widely cultivated.     

Identification of qRBS1, a QTL involved in resistance to bacterial seedling rot in rice

Bacterial seedling rot (BSR), a destructive disease of rice (Oryza sativa L.), is caused by the bacterial pathogen Burkholderia glumae. To identify QTLs for resistance to BSR, we conducted a QTL analysis using chromosome segment substitution lines (CSSLs) derived from a cross between Nona Bokra (resistant) and Koshihikari (susceptible). Comparison of the levels of BSR in the CSSLs and their recurrent parent, Koshihikari, revealed that a region on chromosome 10 was associated with resistance. Further genetic analyses using an F₅ population derived from a cross between a resistant CSSL and Koshihikari confirmed that a QTL for BSR resistance was located on the short arm of chromosome 10. The Nona Bokra allele was associated with resistance to BSR. Substitution mapping in the Koshihikari genetic background demonstrated that the QTL, here designated as qRBS1 (quantitative trait locus for RESISTANCE TO BACTERIAL SEEDLING ROT 1), was located in a 393-kb interval (based on the Nipponbare reference genome sequence) defined by simple sequence repeat markers RM24930 and RM24944.     

Characterization of the promoter controlling Mona/Gads expression in the megakaryocytic lineage

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.     

Genomic map of Clostridium perfringens strain 13

A physical and genetic map of Clostridium perfringens strain 13 was constructed. C. perfringens strain 13 was found to have a 3.1-Mb chromosome and a large 50-kb plasmid, indicating that strain 13 has a relatively small genome among C. perfringens strains. A total of 313 genetic markers were mapped on the chromosome of strain 13. Compared with the physical and genetic map of C. perfringens CPN50, strain 13 had a quite similar genome organization, but with a large deletion (approximately 400 kb) in a particular segment of the chromosome. Among several toxin genes, a beta2 toxin gene that is a novel virulence gene in C. perfringens was found to be located on the 50-kb plasmid.